The long-term objective of this program of research is to develop methods of structural and functional studies for macromolecular assemblies in single-particle (noncrystalline) form, using low-dose electron microscopy of frozen hydrated samples. The recent publication of a 25 angstrom 3D cryo map of the ribosome has been one of the most successful application of these methods to date. Subsequently we were able to use 3D cryo EM difference mapping to locate tRNA in a translating ribosome. The proposed program of research seeks to seize the new opportunities of 3D difference mapping. Specifically, this proposal focuses on the study of the elongation cycle, subunit-subunit association and the path of the nascent polypeptide in the prokaryotic ribosome. We will also begin to use structural and functional probes along with 3D cryo-EM difference mapping in the study of the yeast ribosome. Hand in hand with these investigations, we will continue to make efforts to improve the resolution. To this end we will develop a spot scan data collection procedure, streamline particle selection and processing to enable collection of increased numbers of particles, and work towards an improved understanding of image formation for biological particles in ice. We will also implement time-resolved techniques to study transient states of tRNA, factor binding, and possible conformational changes of the ribosome during the elongation cycle.